Method for isolating proteins or protein and nucleic acid associations, or particle and protein complexes, reagent and uses

ABSTRACT

The invention concerns a method for isolating proteins and/or protein and nucleic acid associations in a sample, comprising steps which consist in: contacting said sample and magnetic colloidal particles comprising a core and a coat wherein: the core is magnetic and is coated with at least a polymer comprising functional groups X selected among amine, hydroxyl, thiol, aldehyde, ester, anhydride, acid chloride, carbonate, carbamate, isocyanate and isothiocyanate groups or mixtures thereof, whereof at least one fraction has reacted with other functional groups of the coat, and the coat consists of a polymer bearing functional groups Z and Z′, capable of ionisation, identical or different, selected among amine, carboxylic acid, ester, anhydride, aldehyde, thiol, disulphide, α-halogenocarbonyl, sulphonic acid, maleimide, isocyanate and isothiocyanate groups to form a mixture; incubating said mixture in predetermined conditions; and separating from the mixture the protein and/or protein and nucleic acid associations complexed on the colloidal particles by applying a magnetic field. The invention also concerns a complex consisting of colloidal particles and proteins, a reagent comprising such a complex or colloidal particles.

[0001] One of the problems which is repeatedly encountered concerns theextraction, purification, concentration and conservation of biologicalmaterial for the purpose of subsequent use.

[0002] One of the protein purification methods used is purification byprecipitation using precipitating agents, such as ammonium sulfate orpoly(acrylic acids) (H. Morawetz and W. L. Hughes, J. Phys. Chem., 56,64-69 (1952)).

[0003] Another method relates to the extraction of biological materialusing colloidal suspensions. Mention may be made of patent applicationWO-A-98/01482 by Krupey, which describes extraction of proteins in anaqueous medium using colloids based on crosslinked maleic anhydridecopolymers. After complexation of the proteins on the colloids, thecomplexes are extracted from the medium by centrifugation. Such a methodhas the drawback of requiring a relatively delicate centrifugation stepand involving considerable and expensive equipment which, in addition,is not always readily accessible.

[0004] A possible solution is to use magnetic iron oxide colloids, asdescribed by VC Rao et al. (Appl. Environ. Microbiol., 1981, Sep.,42(3): 421-426). However, it is known that iron oxides are incompatiblewith techniques for enzymatically amplifying nucleic acids since theyinhibit the enzymes.

[0005] To solve this problem, it has been proposed (patent applicationWO-A-99/35500) to cover magnetic particles with a cationic or anionichydrophilic polymer, which masks the iron oxides and which thus makes itpossible to lift the inhibition of the enzymatic amplification reaction,after a step of extraction of the nucleic acids. This polymer is aheat-sensitive polymer which, when it is heated to a temperature greaterthan 32° C., becomes hydrophobic and can attach proteins via hydrophobicinteractions. However, hydrophobic interactions are denaturing for alarge number of proteins.

[0006] The present inventors have now found magnetic colloidal particleswhich overcome all the abovementioned drawbacks and which are alsoubiquitous, in the sense that they allow the isolation of proteins andof protein and nucleic acid associations from a complex medium in asimple, effective and rapid manner which does not require expensiveequipment, and which are compatible with enzymatic amplificationtechniques. They do not involve extensive equipment, nor manymanipulations. Such particles can therefore be used independently of theenvironment in which they are located and allow, inter alia, theextraction of protein and nucleic acid associations, but also theextraction of proteins.

[0007] The term “proteins” is intended to mean holoproteins andheteroproteins, or fragments thereof, i.e. lipoproteins, glycoproteins,hemoproteins, phosphoproteins, flavoproteins, metalloproteins,polypeptides, antigens, immunogens, antibodies and enzymes.

[0008] The expression “protein and nucleic acid association” is inparticular intended to mean complex nucleoprotein structures such aschromosomes or histones, and also viruses, bacteria and fungi.

[0009] Such particles are particularly advantageous in regions wherehemorrhagic fever viruses are endemic.

[0010] In particular, the particles of the invention may be transportedwithout a lyophilization step or a freezing step, which hasunquestionable advantages, in particular in terms of safety wheninfectious samples are transported. A recent epidemic in the Congoshowed the difficulties which may be encountered in the field, whensamples of human origin which posed very considerable risks ofcontamination were transported to South Africa for diagnosis (WorldHealth Organization, 1999:

[0011] http://www.who.int/emc/outbreak-news/n1999/may/n28may1999.html).

[0012] In addition, the colloidal particles of the invention make itpossible, if desired, to infect cells placed in culture, with aninfectious agent thus isolated, purified, concentrated and, optionally,transported. Thus, there are many applications and uses for thecolloidal particles of the invention.

[0013] In the case of viruses, for example, a step of lysis usingchaotropic agents, heat or other means is essential prior to theenzymatic amplification phase, which requires the use of colloids whichare stable under high stringency conditions. The method for preparingthese stable colloids is incorporated into the examples of the presentpatent application by way of illustration.

[0014] Thus, the present invention relates to a method for isolatingproteins and/or a protein and nucleic acid association, from a sample,characterized in that:

[0015] said sample is brought into contact with magnetic colloidalparticles comprising a core and an envelope in which:

[0016] the core is magnetic and is coated with at least one polymercomprising functional groups X chosen from amine, hydroxyl, thiol,aldehyde, ester, anhydride, acid chloride, carbonate, carbamate,isocyanate and isothiocyanate groups, or mixtures thereof, at least onefraction of which has reacted with other functional groups of theenvelope, and

[0017] the envelope consists of a polymer bearing ionizable functionalgroups, Z and Z′, which may be identical or different, chosen fromamine, carboxylic acid, ester, anhydride, aldehyde, thiol, disulfide,α-halocarbonyl, sulfonic acid, maleimide, isocyanate and isothiocyanategroups, which have partially reacted with the functional groups X of thecore;

[0018] so as to constitute a mixture,

[0019] said mixture is incubated under predetermined conditions, and

[0020] the proteins and/or the protein and nucleic acid associationscomplexed on said colloidal particles are separated from the mixture byapplying a magnetic field.

[0021] In particular, the magnetic core is solid and consists of metaloxide particles, or it consists of an emulsion comprising metal oxideparticles, said metal oxide particles or said emulsion of metal oxideparticles.

[0022] From “partial reaction of the functional groups Z, Z′ with X”, itis understood that groups Z and/or Z′ must remain available. All or atleast some of these free groups Z and/or Z will react via ionicinteraction with the ionic groups of the proteins and/or protein andnucleic acid associations.

[0023] Table 1 below summarizes the complementarity between the variousfunctional groups X, Z and Z′. TABLE 1 X Z Z′ Ester, anhydride, AmineAmine, thiol, acid chloride, carboxylic acid, carbonate, ester, sulfoniccarbamate, acid isocyanate, isothiocyanate Aldehyde Amine Amine, thiol,carboxylic acid, ester, sulfonic acid Amine, hydroxyl Carboxylic acid,Carboxylic acid, ester, anhydride, sulfonic acid aldehyde, isocyanate,isothiocyanate Thiol Thiol, disulfide, Amine, carboxylic α-halocarbonylacid, ester, maleimide anhydride, sulfonic acid

[0024] The core of the colloidal particles comprises at least oneorganic polymer chosen from at least one homopolymer or one copolymer,or mixtures thereof, derived from the polymerization of at least onemonomer chosen from monomers of acrylamide and of acrylate, inparticular N-alkylacrylamides and N,N-dialkyl acrylamides, such asN-isopropylacrylamide, N-methyl acrylamide, N-ethylmethacrylamide,N-n-propyl acrylamide, N-n-propylmethacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N,N-diethylacryl amide,N-methyl-N-isopropylacrylamide or N-methyl-N-n-propylacrylamide;alkylacrylates and alkylmethacrylates in which the alkyl group comprisesfrom 3 to 20 carbon atoms; styrene, methylstyrene, ethylstyrene,tert-butylstyrene, chloromethylstyrene vinyltoluene; derivatives thereofand the copolymers of these monomers with one another and/or with othercomonomers, and metal oxide particles chosen from particles of metaloxides of iron, titanium, cobalt, zinc, copper, manganese, nickel;magnetite; hematite, ferrites such as manganese, nickel ormanganese-zinc ferrites; alloys of cobalt, nickel.

[0025] The envelope polymer is chosen from at least one hydrophilichomopolymer or copolymer chosen from homopolymers or copolymers:

[0026] derived from the polymerization of at least one monomer chosenfrom monomers derived from acrylamide or from methacrylamide; acrylicacid, methacrylic acid; acrylate and methacrylate derivatives;allylamine; styrene derivatives; on the condition, if it is ahomopolymer, that this homopolymer comprises ionizable functionalgroups; in particular copolymers or homopolymers of maleic anhydride andhomopolymers or copolymers of acryloxysuccinimide,

[0027] polysaccharides, such as chitosan and poly(galacturonic acid),

[0028] polypeptides, such as polylysine and polyarginine,

[0029] linear or branched polyethyleneimine, and

[0030] dendrimers;

[0031] preferably poly(maleic anhydride vinyl ether),poly(N-vinylmorpholine-N-acryloxysuccinimide) orpoly-(N-vinylpyrrolidone-N-acryloxysuccinimide).

[0032] The mixture is subjected to incubation at a temperature ofbetween 15 and 60° C., preferably between 20 and 35° C., for anincubation time of between 5 and 60 minutes, preferably 10 minutes.

[0033] The sample may be a biological sample, for example a specimen,such as a tissue specimen, a specimen of whole blood or of serum or aculture supernatant, or alternatively an agrofoods specimen, comprisingproteins and/or protein and nucleic acid associations, in particular avirus, a bacterium, a yeast and/or a cell, optionally as a mixture. The“agrofoods specimen” is intended to mean any agrofoods sample liable tobe, or to have been, infected with an infectious agent.

[0034] The invention also relates to a method for extracting proteinsand/or a protein and nucleic acid association, according to which avirus and/or a bacterium and/or a yeast and/or a cell or a mixturethereof is isolated from a sample, for example an agrofoods specimen ora biological sample such as a specimen or a culture supernatant,according to the method described above and, if necessary, said virus,bacterium, yeast, cell or mixture thereof is subjected to a step ofpartial or total release and/or denaturation for the extraction of saidproteins and/or said nucleic acids.

[0035] The invention also relates to a method for identifying and/ordetecting and/or quantifying proteins and/or a protein and nucleic acidassociation and/or nucleic acids, according to which a virus and/or abacterium and/or a yeast and/or a cell or a mixture thereof is isolatedfrom a sample, for example from a specimen or from a culturesupernatant, according to the method described above, if necessary saidvirus and/or bacterium and/or yeast and/or cell or mixture thereof issubjected to a step of partial or total release and/or denaturation forthe extraction of said proteins and/or said nucleic acids, and saidproteins are identified and/or detected and/or quantified by immunoassayand/or said nucleic acids are identified and/or detected and/orquantified by amplifying them and/or by hybridization of at least onenucleotide probe specific for said nucleic acids to be identified and/ordetected and/or quantified.

[0036] The term “immunoassay” is intended to mean the detection and/orquantification of at least one protein or of at least one antigen byrevelation of a protein or antigen/antibody complex, in particular using“Western blotting”, “competition” or “sandwich” techniques.

[0037] The term “amplification” is intended to mean all techniquessuitable for amplifying DNA or RNA, in particular PCR, RT-PCR, NASBA,and TMA. It may involve, inter alia, a quantitative amplification.Moreover, the nucleic acids to be identified and/or detected and/orquantified may be so by hybridization of at least one nucleotide probelabeled with any suitable label, preferably by hybridization of thetarget to a capture probe and to a detection probe. By way ofnonlimiting example, mention may be made of the techniques, which arewell known to those skilled in the art, of Southern blotting and ofNorthern blotting and of the ELOSA (Enzyme Linked Oligosorbent Assay)technique (Katz JB et al., Am. J. Vet. Res., December 1993; 54 (12):2021-6 and Francois Mallet et al., Journal of Clinical Microbiology,June 1993, p. 1444-1449).

[0038] The proteins are surface or intracellular proteins of a virus, ofa bacterium, of a yeast or of a cell, and the nucleic acids are DNAand/or RNA. The proteins are identified and/or detected and/orquantified (i) either directly, without a step of release and/ordenaturation, from said virus and/or bacterium and/or yeast and/or cell,isolated or in a culture supernatant, (ii) or indirectly, after a stepof partial or total release and/or denaturation carried out, forexample, by modification of pH (decrease or increase in pH by additionof an acid or basic solution). The nucleic acids are identified and/ordetected and/or quantified after a step of partial or total releaseand/or denaturation of said isolated virus and/or bacterium and/or yeastand/or cell carried out, for example, by chemical and/or physicaltreatment. By way of example, a chemical treatment includes the use ofsurfactants, such as SDS or LLS, the use of chaotropic agents, such asguanidium thiocyanate, and the use of enzymes, such as proteases(proteinase K), or other suitable agents. By way of example, a physicaltreatment comprises sonication, heating, ultrasound, mechanicalagitation (with, for example, rigid beads of the glass type) or thelike.

[0039] The invention also relates to a method for culturing a virusand/or a bacterium and/or a yeast and/or cells, according to which saidvirus and/or said bacterium and/or said yeast and/or said cells areisolated from a sample, for example a specimen or a culture supernatant,according to the method described above, said virus and/or bacteriumand/or yeast and/or cells thus isolated are placed in culture, in aculture medium and under conditions which are suitable, and, if desired,said virus and/or bacterium and/or yeast and/or cells are identifiedand/or detected and/or quantified according to techniques well known tothose skilled in the art.

[0040] Another subject of the invention is a method for preparing abiological sample, such as a specimen or a culture supernatant, or anagrofoods specimen, according to which proteins and/or protein andnucleic acid associations are isolated from the sample, according to themethod above, and/or a culture is prepared according to a method asdescribed above.

[0041] The invention also relates to a complex made up of magneticcolloidal particles and proteins and/or a protein and nucleic acidassociation, the proteins and nucleic acids being immobilized byelectrostatic interactions and/or by adsorption. The colloidal particlescomprise a core and an envelope in which:

[0042] the core is magnetic and is coated with at least one polymercomprising functional groups X chosen from amine, hydroxyl, thiol,aldehyde, ester, anhydride, acid chloride, carbonate, carbamate,isocyanate and isothiocyanate groups, or mixtures thereof, at least onefraction of which has reacted with other functional groups of theenvelope, and

[0043] the envelope consists of a polymer bearing ionizable functionalgroups, Z and Z′, which may be identical or different, chosen fromamine, carboxylic acid, ester, anhydride, aldehyde, thiol, disulfide,α-halocarbonyl, sulfonic acid, maleimide, isocyanate and isothiocyanategroups, at least some of which have reacted with the functional groups Xof the core; and

[0044] the proteins and/or protein and nucleic acid associations arederived from a biological sample, for example from a specimen, such as atissue specimen or a specimen of whole blood or of serum, or from aculture supernatant, or from an agrofoods specimen.

[0045] In particular, the core of the colloidal particles is solid andconsists of metal oxide particles, or is essentially solid and consistsof an emulsion comprising metal oxide particles.

[0046] Preferably, the specimen or the culture supernatant is infectedwith at least one virus, one bacterium or one yeast, or a mixturethereof.

[0047] The core comprises at least one organic polymer chosen from atleast one homopolymer or one copolymer, or mixtures thereof, derivedfrom the polymerization of at least one monomer chosen from monomers ofacrylamide and of acrylate, in particular N-alkylacrylamides andN,N-dialkylacrylamides, such as N-isopropylacrylamide,N-methylacrylamide, N-ethylmethacrylamide, N-n-propyl acrylamide,N-n-propylmethacrylamide, N-isopropylmeth acrylamide,N-cyclopropylacrylamide, N,N-diethylacryl amide,N-methyl-N-isopropylacrylamide or N-methyl-N-n-propylacrylamide;alkylacrylates and alkylmethacrylates in which the alkyl group comprisesfrom 3 to 20 carbon atoms; styrene, methylstyrene, ethylstyrene,tert-butylstyrene, chloromethylstyrene vinyltoluene; derivatives thereofand the copolymers of these monomers with one another and/or with othercomonomers, and metal oxide particles chosen from particles of metaloxides of iron, titanium, cobalt, zinc, copper, manganese, nickel;magnetite; hematite, ferrites such as manganese, nickel ormanganese-zinc ferrites; alloys of cobalt, nickel.

[0048] The envelope polymer is chosen from at least one hydrophilichomopolymer or copolymer chosen from homopolymers or copolymers:

[0049] derived from the polymerization of at least one monomer chosenfrom monomers derived from acrylamide or from methacrylamide; acrylicacid, methacrylic acid; acrylate and methacrylate derivatives;allylamine; styrene derivatives; on the condition, if it is ahomopolymer, that this homopolymer comprises ionizable functionalgroups; in particular copolymers or homopolymers of maleic anhydride andhomopolymers or copolymers of acryloxysuccinimide,

[0050] polysaccharides, such as chitosan and poly(galacturonic acid),

[0051] polypeptides, such as polylysine and polyarginine,

[0052] linear or branched polyethyleneimine, and

[0053] dendrimers;

[0054] in particular poly(maleic anhydride vinyl ether),poly(N-vinylmorpholine-N-acryloxysuccinimide) orpoly(N-vinylpyrrolidone-N-acryloxysuccinimide).

[0055] This complex is used for the transfer and/or transport and/orstorage of infectious agents, in particular of a virus and/or bacteriumand/or yeast, in the dry state or in a suitable buffer.

[0056] The invention also relates to a reagent for extracting and/oridentifying and/or detecting and/or quantifying proteins and/or proteinand nucleic acid associations, characterized in that it comprises, interalia:

[0057] a complex as defined above, or colloidal particles as definedabove which make it possible to obtain a complex of the invention, and

[0058] optionally at least one means for identifying and/or detectingand/or quantifying said proteins or said nucleic acids, in particular atleast one monoclonal or polyclonal antibody for identifying and/ordetecting and/or quantifying at least one protein, and at least oneprimer, preferably at least two primers, and/or at least one nucleotideprobe, preferably at least two nucleotide probes, specific for at leastone nucleic acid, for the identification and/or detection and/orquantification thereof.

[0059] Finally, the invention relates to a vaccinal composition whichcomprises, as active principle, at least one complex of the invention asdefined above, optionally combined with a pharmaceutically acceptablevehicle and/or excipient and/or adjuvant and/or diluent.

[0060] The term “pharmaceutically acceptable vehicle” is intended tomean the carriers and vehicles which can be administered to humans or toanimals, as described, for example, in Remington's PharmaceuticalSciences 16th ed., Mack Publishing Co. The pharmaceutically acceptablevehicle is preferably isotonic or hypotonic or is weakly hypertonic andhas a relatively low ionic strength. The definitions of thepharmaceutically acceptable excipients and adjuvants are also given inRemington's Pharmaceutical Sciences, mentioned above.

[0061] Another subject of the invention is a pharmaceutically acceptablevehicle for a vaccinal composition, consisting of a magnetic particlecorresponding to the definition of the present invention.

EXAMPLE 1

[0062] Production of Carboxylic Magnetic Latexes

[0063] The polymer poly(maleic anhydride methyl vinyl ether) (MAMVE) issolubilized in anhydrous dimethyl sulfoxide (DMSO) (2 g/l). 50 μl ofthis MAMVE solution are diluted in 1 ml of phosphate buffer (pH 6.8; 10mM) and then incubated for 10 minutes at 37° C. 1 ml of a dispersion ofaminated magnetic latex (0.5% in water, 1× the critical micellarconcentration (CMC) of Triton X-405) produced, for example, according tothe protocol described in patent application PCT WO 99/35500, is thenmixed with 125 μl of the previously prepared mixture(MAMVE-DMSO-buffer). The mixture is incubated at 37° C. for 3 hours. Theparticles can then be solubilized as they are or after a purificationstep, for example by centrifugation.

EXAMPLE 2

[0064] Selective Extraction of Viral Particles from Serum, from Plasmaor from Culture Supernatants.

[0065] Purification of viral particles:

[0066] From 100 to 150 μl of positive serum, i.e. serum comprising viralparticles, are added directly to 5 μl of magnetic latex functionalizedwith the MAMVE copolymer, as described in Example 1, at a solids contentof 3%. The sample is not buffered.

[0067] The serum-latex mixture is homogenized on a vortex for 10 secondsand then left at ambient temperature for 10 minutes in order for themagnetic latex to attach the viral particles. This capture step isfollowed by recovery of the latex-viral particle complex, in the form ofa pellet from which the supernatant has been removed, by simplemagnetization for 30 seconds.

[0068] The pellet may or may not be washed once with 100 μl of Hepes, pH6.5, magnetized as previously and then dispersed in 50 μl of sterilewater.

[0069] Extraction of nucleic acids after heat-lysis of viral particles:

[0070] The latex-viral particle complex dispersed in water is incubatedat 95° C. for 5 minutes in order to lyse the viral particles. Thenucleic acids are recovered from the supernatant from which the latexhas been removed by magnetization.

EXAMPLE 3

[0071] Capture of the Hepatitis G Virus using a Functionalized MagneticLatex of the Invention.

[0072] Purification of viral particles:

[0073] Increasing volumes, respectively of 25 μl and 50 μl, of undilutedhuman serum positive for the hepatitis G virus, the viral titer of whichis unknown, are added to 5 μl of the magnetic latex functionalized withthe MAMVE copolymer, as described in Example 1, at a solids content of3%.

[0074] The serum-latex mixture is homogenized on a vortex for 10 secondsand then left at ambient temperature for 10 minutes in order for themagnetic latex to attach the viral particles. This capture step isfollowed by recovery of the latex-viral particle complex, in the form ofa pellet from which the supernatant has been removed, by simplemagnetization for 5 minutes.

[0075] The pellet is then washed once with 50 μl of Hepes-Tween (0.25/∞,pH 6.5), magnetized as previously, and then dispersed in 50 μl ofsterile water.

[0076] Extraction of nucleic acids:

[0077] A step of viral particle lysis is then carried out using theQIAmp Viral RNA Mini Kit (commercial name), marketed by the companyQIAGEN, so as to release the viral nucleic acids.

[0078] Amplification of nucleic acids:

[0079] The viral nucleic acids are then amplified by one-step RT-PCR (1)(Life Technologies) and nested PCR (2) using the following pairs ofprimers (Smith DB et al., Discrimination of hepatitis G virus/GBV-Cgeographical variants by analysis of the 5′ non-coding region, J. Gen.Virol., 1997, 78: 1533-1542): (1) primer 1: 5′AGG TGT GGA TGG GTG ATG 3′    primer 2: 5′ATG CCA CCC GCC CTC ACC CGA 3′ (2) primer 1: 5′TTG GTAGGT CGT AAA TCC CGG 3′    primer 2: 5′CGG AGC TGG GTG GCC CCA TGC ATT 3′

[0080] The results, after running on a 1% agarose gel and staining withethidium bromide, show a band of expected size of 343 base pairs, afterthe second PCR. The molecular weight marker used as reference is theSmartladder marker (commercial name) (multiples of 200 base pairs) fromthe company Eurogentec. These results confirm that the functionalizedlatex of the invention is capable of capturing the hepatitis G viralparticles and of retaining them during the washing steps.

[0081] Moreover, 10 μl of human serum positive for HGV, of unknowntiter, were diluted in 3 ml of negative human serum and brought intocontact with 10 μl of the latex of the invention, obtained according toExample 1. The viral particle purification, nucleic acid extraction andamplification steps were carried out as described above.

[0082] The results obtained show, after running the amplificationproducts on 1% agarose gel and staining with ethidium bromide, the bandof expected size of 343 base pairs, which confirms the ability of thelatex of the invention to capture and retain the viral particles from adiluted sample of large volume.

EXAMPLE 4

[0083] Capture of the hepatitis C virus with a functionalized magneticlatex of the invention.

[0084] Purification of viral particles:

[0085] 50 μl volumes of an undiluted human serum positive for thehepatitis C virus, the viral titer of which is unknown, are added to 5μl of the magnetic latex functionalized with the MAMVE copolymer, asdescribed in Example 1, at a solids content of 3%.

[0086] The serum-latex mixture is homogenized on a vortex for 10 secondsand then left at ambient temperature for 10 minutes in order for themagnetic latex to attach the viral particles. This capture step isfollowed by recovery of the latex-viral particle complex, in the form ofa pellet from which the supernatant has been removed, by simplemagnetization for 5 minutes.

[0087] The pellet is then washed once with 50 μl of Hepes-Tween (0.25/∞,pH 6.5), magnetized as previously, and then dispersed in 50 μl ofsterile water.

[0088] Extraction of nucleic acids:

[0089] A step of nucleic acid extraction is then carried out using theQIAmp Viral RNA Mini Kit (commercial name), marketed by the companyQIAGEN, so as to release the viral nucleic acids.

[0090] Amplification of nucleic acids:

[0091] The viral nucleic acids are then amplified by one-step RT-PCR (1)(Life Technologies), followed by a semi-nested PCR (2) using thefollowing pairs of primers (Li JS et al., Identification of the thirdmajor genotype of hepatitis C in France, BBRC, 1994, 3: 1474-1481): (1):primer 1: 5′ CCT GTG AGG AAC TAC TGT CTT CAC GCA 3′      primer 2:5′ ACT CGC AAG CAC CCT ATC AGG CAG TAC 3′ (2): primer 1: 5′ AAG CGT CTAGCC ATG GCG TTA GTA T 3′      primer 2: 5′ ACT CGC AAG CAC CCT ATC AGGCAG TAC 3′

[0092] The results, after running on a 1% agarose gel and staining withethidium bromide, show a band of expected size of 240 base pairs, afterthe second PCR. The molecular weight marker used as reference is theSmartladder marker (commercial name) (multiples of 200 base pairs) fromthe company Eurogentec. These results confirm that the functionalizedlatex of the invention is capable of capturing the hepatitis G viralparticles and of retaining them during the washing steps.

EXAMPLE 5

[0093] Detection of the Measles Virus.

[0094] The nucleic acids were extracted from previously titered viralsamples using the kit marketed by the company QIAGEN (QIAmp Viral RNAMini Kit (commercial name)), according to the supplier's protocol. Thenucleic acids were then amplified by RT-PCR using the TITAN kit(commercial name—Roche) in the presence of 1 μl of RNAase inhibitor(RNAsin, commercial name—Promega) or by nested PCR.

[0095] Titering of viral samples (Edmonston strain): successivedilutions of the virus were prepared (from 10⁻¹ to 10⁻⁷). 200 μl ofthese dilutions were distributed into the 6 wells of a plate containingVero cells (green monkey kidney cells) cultured in a medium 199containing glutamax (Gibco-BRL), 1% of antibiotics (Gibco-BRL) and 1% offetal calf serum. The Vero cells are 80% confluent. Incubation wascarried out for one hour at 37° C. under CO₂ (5%). 200 μl of the mediumcontaining the virus were removed and replaced with 200 μl of virus-freemedium. Incubation was carried out under the same conditions for 72 to96 hours. The cells are then examined and the lysis plaques arevisualized and counted. 60 ml of culture with a titer of 5.5 10⁶ pfu/ml(lysis plaque forming unit/ml) were obtained, aliquoted and stored at↑80° C.

[0096] Extraction of nucleic acids: the nucleic acids are extracted fromthe culture (5.5 10³ pfu/μl) using the abovementioned kit from thecompany QIAGEN.

[0097] Amplifications: the extracted nucleic acids are amplified byRT-PCR using the following pair of PCR primers: primer 1: 5′ CCC ATT ACATCA GGA TCC GG 3′ primer 2: 5′ TTG GTC CGC CTC ATC CTC CA 3′

[0098] optionally followed by a nested PCR using the following pair ofprimers: primer 1: 5′ GGT ACC TCT TGA TGC GAA GG 3′ primer 2: 5′ GGC CACACT TTT AAG GAG CTT A 3′

[0099] The amplification products are then run on 2% agarose gel(TBE+Gelstar). The molecular weight marker used as reference is theSmartladder marker (commercial name) (multiples of 100 base pairs) fromthe company Eurogentec. The results show a band of expected size of 384base pairs for the RT-PCR and of 309 base pairs for the nested PCR,which confirms the correct extraction of the viral RNAs and theefficiency of both the RT-PCR and the nested PCR.

EXAMPLE 6

[0100] Capture of the Measles Virus using the Functionalized Latex ofthe Invention.

[0101] After obtaining titered viral samples, as described in Example 8,the level of viral capture and detection using the latex of Example 1was quantitatively determined. A range of viral titers was produced bydiluting the stock solution in negative human plasma. 10-fold dilutionswere made in a final volume of 1000 μl. The amount of latex is 35 μl(945 μg) per sample. After capture, the viral RNAs are extracted usingthe abovementioned QIAGEN kit. The extracted viral RNAs are thenamplified by RT-PCR followed by a nested PCR using the pairs of primersdescribed in Example 5. The amplification products are then run on a 2%agarose gel (TBE+Gelstar). A molecular weight marker (multiple of 200base pairs) (Smartladder-Eurogentec) is used as reference. The positivecontrol is represented by the extracted total RNA and the negativecontrol consists of negative plasma. The results are given in Table 2below. TABLE 2 Negative Positive control control Dilutions in pfu (lysisplaque forming Negative Total unit) plasma RNA 10⁶ 10⁵ 10⁴ 10³ 10² 10 110⁻¹ − + + + + + + + + −

[0102] The symbol+signifies the presence of the amplification product ofexpected size (309 base pairs) and the symbol—signifies an absence ofamplification product.

[0103] The results show that the nested PCR gives a specificamplification product down to 1 pfu per tube (100 μl). The absence ofamplification product at the 0.1 pfu dilution indicates that there is nodetection of noninfectious viral particles.

Example 7

[0104] Study of the Stability of the Latex/measles Virus Complex.

[0105] In order to study the stability of the latex/virus complexes,relative to the detection of the viral RNAs by RT-PCR, the complexeswere revealed over time. Several infectious titers were tested, as weredifferent treatments of the samples, with the aim of preserving thesamples while at the same time reducing the number of manipulations. Thesamples were kept under a laminar flow hood, at ambient temperature.

[0106] The complexes, having undergone treatments or not havingundergone treatments, were tested on day 0, 1, 2, 3, 7 and 14,respectively:

[0107] Treatment A: sample of plasma containing the virus and the latex,which serves as a basic control (no treatment).

[0108] Treatment B: latex/virus complex, after removal of the plasma, nowashing and left dry.

[0109] Treatment C: latex/virus complex, after removal of the plasma anda washing step and left dry.

[0110] Treatment D: latex/virus complex, after removal of the plasma anda washing step and kept in the presence of washing buffer.

[0111] The infectious titers of the samples tested are, respectively,50, 500 and 5000 pfu per sample for each treatment.

[0112] The complexes are then heat-lysed so as to release the nucleicacids, and the nucleic acids are run on 2% agarose gels (TBE+Gelstar)with a molecular weight marker used as reference (Smartladder-Eurogentec).

[0113] The results, under the conditions of the experiment, show thattreatment B does not make it possible to conserve the viral particles ina stable manner. However, it should be noted that, if a washing step iscarried out before the nucleic acid extraction step, the latex of theinvention conserves the particles in a stable manner, as shown by theinventors in another experiment. On the other hand, complexes C and D,which undergo a washing step, make it possible to conserve the sampleswithout the RNAs being degraded, as demonstrated by RT-PCR. This meansthat a step consisting of washing in a buffer is necessary in order toconserve the captured sample, whether the latter is conserved in thebuffer or dry, if the intention is to carry out the lysis step in theextraction tube, but a means of avoiding this washing step is to changetube. Of course, it is more advantageous to conserve the capturedsamples dry, since this makes it possible to transport them from theplace where they are taken to a site for analysis while avoiding anyrisk of contamination and of dissemination.

Example 8

[0114] Capture of the Measles Virus from Whole Blood.

[0115] Tests were carried out using whole blood. A range of virus wasestablished, in whole human blood and plasma of the same origin, with anequal amount of latex. The latex/viral particle complexes were subjectedto heat treatment in order to extract the nucleic acids. The nucleicacids released were amplified by RT-PCR using the primers previouslydescribed.

[0116] The results obtained after running the amplification products on2% agarose gel (TBE+Gelstar) show a band of expected size of 384 basepairs starting from a viral titer of 5 10³ pfu in the whole blood andfrom 50 pfu in the plasma. These results show that it is possible toisolate viral particles from whole blood, although this is substantiallyless efficient than from plasma. The results are summarized in Table 3below. TABLE 3 Infectious titer in pfu Sample 0 10 50 500 5000 50 000Blood − − − − + + Plasma − − + + + +

EXAMPLE 9

[0117] Culturing of Captured Measles Viruses.

[0118] Using human plasma, a viral range was established and tested asdescribed in Example 8 (see Table 2). The magnetic latex/measles viruscomplexes were placed in culture in the presence of Vero cells culturedin a medium 199 containing glutamax (Gibco-BRL), 1% of antibiotics(GIBCO-BRL) and 10% of fetal calf serum. The culturing conditions wereestablished as described in Example 5. An RT-PCR was carried out on theviral range, the very day on which it was placed in culture, so as tovalidate the presence of the virus in the sample placed in culture. Thecells of the culture were then observed under a microscope so as todetect a cytopathic effect induced by the infectious virus in theculture. A cytopathic effect was observed at the end of the third dayfor the samples for which the infectious titer was 5 10⁴ and 5 10³pfu/ml, and after 5 days for the samples for which the infectious titerwas 500 and 50 pfu/ml.

EXAMPLE 10

[0119] Capture of the Acquired Immunodeficiency Syndrome Virus (HIV-1)using the Functionalized Latex of the Invention, from a CultureSupernatant, and Study of the Viral Viability.

[0120] Viral capture:

[0121] An HIV-1 viral strain of group M, subtype G was cultured in thepresence of PBMCs (peripheral blood mononuclear cells) in an RPMIculture medium supplemented with 10% of fetal calf serum. The presenceof the virus was confirmed on the 14th day of culturing by detection ofthe P24 iantigen (31637.8 pg/ml, dilution to 1/100) in the culturesupernatant using the VIDAS HIV P24 II immunoenzymatic test carried outon the VIDAS machine (registered trademark—bioMérieux). 150 μl of thisculture supernatant were brought into contact with 10 μl of magneticlatex, obtained as described in Example 1, for 10 minutes at ambienttemperature and with stirring. The capture step was followed by amagnetization step in order to separate the pellet from the culturesupernatant, and the pellet thus obtained (latex-virus complex) waswashed with approximately 100 μl of RPMI culture medium, and thenmagnetized again and resuspended in 10 μl of RPMI medium.

[0122] Viability:

[0123] 10 μl of the latex-virus complex prepared as described above werebrought into contact with a pellet of PBMCs (25 10⁶ cells), freshlyprepared. The mixture was homogenized and then subjected to incubationat 37° C. for two hours, for the PBMC infection step, under anatmosphere of CO₂ (5%). 15 ml of RPMI medium were then added. Themixture was homogenized and 5 ml of medium were removed on DO andcentrifuged at 1100 rpm for 10 minutes. 1 ml of supernatant will be usedto assay the P24 and the remainder is returned to the correspondingculture medium. In parallel, freshly prepared PBMCs were placed inculture, under the same conditions, in the presence of 10 μl of latexalone (without virus) so as to constitute the negative control. The P24antigen is assayed, using the immunoenzymatic test (VIDAS HIV P24 II) onthe VIDAS machine, in the control and infected culture supernatant,respectively on days D0, D2, D4, D7, D9, D11, D14, D16 and D18. Theresults obtained show a significant increase in the P24 antigen in theculture supernatant from the 14th day of culturing, which confirms,firstly, that the virus has indeed been captured by the latex of theinvention and that, secondly, it conserves, after capture, its viabilityand its replicative power (infectiousness).

Example 11

[0124] Capture of the Hepatitis B Virus (HBV).

[0125] Ten-fold dilutions of an HBV-positive serum were made, in a finalvolume of 150 μl, containing 10,000 to 10 viral copies. The viralcapture and the viral DNA extraction were carried out according to theprotocol described in Example 2.

[0126] The number of viral copies was detected by nested PCR usingprimers defined in the S region of the HBV genome for the amplificationof a 440 base pair fragment. 1st round: Primer 1: 5′ CCT GCT GGT GGC TCCAGT TC 3′ Primer 2: 5′ TAC CCA AAG ACA AAA GAA AAT TGG 3′ 2nd round:Primer 3: 5′ TAG TAA ACT GAG CCA RGA GAA AC 3′ Primer 4: 5′ GTT GAC AARAAT CCT CAC AAT AC 3′

[0127] The amplification products are then run on a 1% agarose gel andstained with ethidium bromide. A signal is detected at the 10⁻³ dilutioncorresponding to a detection threshold of 10 HBV copies, representativeof good sensitivity of the capture system of the invention.

[0128] Moreover, a positive human serum was diluted in negative humanserum so as to obtain 100 copies of HBV in two different volumes of 100and 1000 μl, in order to study a potential effect of concentration ofthe latex of the invention. After capture of the viral particles,contained in the two diluted samples, using 10 μl of latex, the nucleicacids were extracted by heat lysis and amplified by nested PCR using theprimers described above. The amplification products are then run on 1%agarose gel and stained with ethidium bromide. The results show, for theproducts amplified from the two diluted samples, a band at the expectedsize of 440 base pairs. The intensity of staining of the two fragmentsamplified, derived, respectively, from the abovementioned two dilutedsamples, proves to be identical, which proves an effect of concentrationof the magnetic latex of the invention.

1 16 1 18 DNA Artificial Sequence PCR primer 1 aggtgtggat gggtgatg 18 221 DNA Artificial Sequence PCR primer 2 atgccacccg ccctcacccg a 21 3 21DNA Artificial Sequence PCR primer 3 ttggtaggtc gtaaatcccg g 21 4 24 DNAArtificial Sequence PCR primer 4 cggagctggg tggccccatg catt 24 5 27 DNAArtificial Sequence PCR primer 5 cctgtgagga actactgtct tcacgca 27 6 27DNA Artificial Sequence PCR primer 6 actcgcaagc accctatcag gcagtac 27 725 DNA Artificial Sequence PCR primer 7 aagcgtctag ccatggcgtt agtat 25 827 DNA Artificial Sequence PCR primer 8 actcgcaagc accctatcag gcagtac 279 20 DNA Artificial Sequence PCR primer 9 cccattacat caggatccgg 20 10 20DNA Artificial Sequence PCR primer 10 ttggtccgcc tcatcctcca 20 11 20 DNAArtificial Sequence PCR primer 11 ggtacctctt gatgcgaagg 20 12 22 DNAArtificial Sequence PCR primer 12 ggccacactt ttaaggagct ta 22 13 20 DNAArtificial Sequence PCR primer 13 cctgctggtg gctccagttc 20 14 24 DNAArtificial Sequence PCR primer 14 tacccaaaga caaaagaaaa ttgg 24 15 23DNA Artificial Sequence PCR primer 15 tagtaaactg agccargaga aac 23 16 23DNA Artificial Sequence PCR primer 16 gttgacaara atcctcacaa tac 23

1. A method for isolating proteins and/or a protein and nucleic acidassociation, from a sample, characterized in that: said sample isbrought into contact with magnetic colloidal particles comprising a coreand an envelope in which: the core is magnetic and is coated with atleast one polymer comprising functional groups X chosen from amine,hydroxyl, thiol, aldehyde, ester, anhydride, acid chloride, carbonate,carbamate, isocyanate and isothiocyanate groups, or mixtures thereof, atleast one fraction of which has reacted with other functional groups ofthe envelope, and the envelope consists of a polymer bearing ionizablefunctional groups, Z and Z′, which may be identical or different, chosenfrom amine, carboxylic acid, ester, anhydride, aldehyde, thiol,disulfide, α-halocarbonyl, sulfonic acid, maleimide, isocyanate andisothiocyanate groups, which have partially reacted with the functionalgroups X of the core; so as to constitute a mixture, said mixture isincubated under predetermined conditions, and the proteins and/or theprotein and nucleic acid associations complexed on said colloidalparticles are separated from the mixture by applying a magnetic field.2. The method as claimed in claim 1, characterized in that the corecomprises at least one organic polymer chosen from at least onehomopolymer or one copolymer, or mixtures thereof, derived from thepolymerization of at least one monomer chosen from monomers ofacrylamide and of acrylate, in particular N-alkylacrylamides andN,N-dialkylacrylamides, such as N-isopropylacrylamide,N-methylacrylamide, N-ethylmethacrylamide, N-n-propylacrylamide,N-n-propylmethacrylamide, N-isopropylmethacrylamide,N-cyclopropylacrylamide, N,N-diethylacryl amide,N-methyl-N-isopropylacrylamide or N-methyl-N-n-propylacrylamide;alkylacrylates and alkylmethacrylates in which the alkyl group comprisesfrom 3 to 20 carbon atoms; styrene, methylstyrene, ethylstyrene,tert-butylstyrene, chloromethylstyrene vinyltoluene; derivatives thereofand the copolymers of these monomers with one another and/or with othercomonomers, and metal oxide particles chosen from particles of metaloxides of iron, titanium, cobalt, zinc, copper, manganese, nickel;magnetite; hematite, ferrites such as manganese, nickel ormanganese-zinc ferrites; alloys of cobalt, nickel.
 3. The method asclaimed in claim 1, characterized in that the envelope polymer is chosenfrom at least one hydrophilic homopolymer or copolymer chosen fromhomopolymers or copolymers: derived from the polymerization of at leastone monomer chosen from monomers derived from acrylamide or frommethacrylamide; acrylic acid, methacrylic acid; acrylate andmethacrylate derivatives; allylamine; styrene derivatives; on thecondition, if it is a homopolymer, that this homopolymer comprisesionizable functional groups; in particular copolymers or homopolymers ofmaleic anhydride and homopolymers or copolymers of acryloxysuccinimide,polysaccharides, such as chitosan and poly(galacturonic acid),polypeptides, such as polylysine and polyarginine, linear or branchedpolyethyleneimine, and dendrimers.
 4. The method as claimed in claim 3,characterized in that the envelope polymer is poly(maleic anhydridevinyl ether), poly(N-vinylmorpholine-N-acryloxysuccinimide) orpoly(N-vinylpyrrolidone-N-acryloxysuccinimide).
 5. The method as claimedin any one of the preceding claims, characterized in that the mixture issubjected to incubation at a temperature of between 15 and 60° C.,preferably between 20 and 35° C., for an incubation time of between 5and 60 minutes, preferably 10 minutes.
 6. The method as claimed in anyone of the preceding claims, characterized in that the sample is aspecimen or a culture supernatant comprising proteins and/or protein andnucleic acid associations, in particular a virus, a bacterium, a yeastand/or a cell, optionally as a mixture.
 7. The method as claimed inclaim 6, characterized in that the sample is a biological sample, suchas a tissue specimen, a specimen of whole blood, of plasma or of serumor a culture supernatant, or an agrofoods specimen.
 8. A method forextracting proteins and/or nucleic acids, characterized in that: a virusand/or a bacterium and/or a yeast and/or a cell or a mixture thereof isisolated from a sample, for example from a specimen or from a culturesupernatant, according to the method described in claims 1 to 7, and ifnecessary, said virus, bacterium, yeast, cell or mixture thereof issubjected to a step of partial or total release and/or denaturation forthe extraction of said proteins and/or said nucleic acids.
 9. A methodfor identifying and/or detecting and/or quantifying proteins and/or aprotein and nucleic acid association and/or nucleic acids, characterizedin that: a virus and/or a bacterium and/or a yeast and/or a cell or amixture thereof is isolated from a sample, for example from a specimenor from a culture supernatant, according to the method described inclaims 1 to 7, and if necessary, said virus and/or bacterium and/oryeast and/or cell or mixture thereof is subjected to a step of partialor total release and/or denaturation for the extraction of said proteinsand/or said nucleic acids, and said proteins are identified and/ordetected and/or quantified by immunoassay and/or said nucleic acids areidentified and/or detected and/or quantified by amplifying them and/orby hybridization of at least one nucleotide probe specific for saidnucleic acids to be identified and/or detected and/or quantified. 10.The method as claimed in claim 9, characterized in that the proteins aresurface or intracellular proteins of a virus, of a bacterium, of a yeastor of a cell, and in that the nucleic acids are DNA and/or RNA.
 11. Themethod as claimed in claims 9 and 10, characterized in that the proteinsare identified and/or detected and/or quantified (i) either directly,without a step of release and/or denaturation, from said virus and/orbacterium and/or yeast and/or cell, isolated or in a culturesupernatant, (ii) or indirectly, after a step of partial or totalrelease and/or denaturation carried out, for example, by modification ofpH.
 12. The method as claimed in claims 9 and 10, characterized in thatthe nucleic acids are identified and/or detected and/or quantified aftera step of partial or total release and/or denaturation of said isolatedvirus and/or bacterium and/or yeast and/or cell carried out, forexample, by chemical and/or physical treatment.
 13. A method forculturing a virus and/or a bacterium and/or a yeast and/or cells,characterized in that: said virus and/or said bacterium and/or saidyeast and/or said cells are isolated from a sample, according to themethod described in claims 1 to 7, said virus and/or bacterium and/oryeast and/or cells thus isolated are placed in culture, in a culturemedium and under conditions which are suitable, and, if desired, saidvirus and/or bacterium and/or yeast and/or cells are identified and/ordetected and/or quantified.
 14. A method for preparing a biologicalsample, characterized in that: proteins and/or protein and nucleic acidassociations are isolated from the sample, according to the methoddescribed in claims 1 to 7, and/or a culture is prepared as described inclaim
 13. 15. A complex, characterized in that it is made up of magneticcolloidal particles and proteins and/or a protein and nucleic acidassociation, said colloidal particles comprising a core and an envelopein which: the core is magnetic and is coated with at least one polymercomprising functional groups X chosen from amine, hydroxyl, thiol,aldehyde, ester, anhydride, acid chloride, carbonate, carbamate,isocyanate and isothiocyanate groups, or mixtures thereof, at least onefraction of which has reacted with other functional groups of theenvelope, and the envelope consists of a polymer bearing ionizablefunctional groups, Z and Z′, which may be identical or different, chosenfrom amine, carboxylic acid, ester, anhydride, aldehyde, thiol,disulfide, α-halocarbonyl, sulfonic acid, maleimide, isocyanate andisothiocyanate groups, some of which have reacted with the functionalgroups X of the core; and said proteins and/or protein and nucleic acidassociations being derived from a biological sample, for example from aspecimen, such as a tissue specimen or a specimen of whole blood or ofserum, or from a culture supernatant, or from an agrofoods specimen. 16.The complex as claimed in claim 15, characterized in that the specimenor the culture supernatant is infected with at least one virus, onebacterium or one yeast, or a mixture thereof.
 17. The complex as claimedin claims 15 and 16, characterized in that the core comprises at leastone organic polymer chosen from at least one homopolymer or onecopolymer, or mixtures thereof, derived from the polymerization of atleast one monomer chosen from monomers of acrylamide and of acrylate, inparticular N-alkylacrylamides and N,N-dialkylacrylamides, such asN-isopropylacrylamide, N-methylacrylamide, N-ethyl methacrylamide,N-n-propylacrylamide, N-n-propyl methacrylamide,N-isopropylmethacrylamide, N-cyclo propylacrylamide,N,N-diethylacrylamide, N-methyl-N isopropylacrylamide orN-methyl-N-n-propylacrylamide; alkylacrylates and alkylmethacrylates inwhich the alkyl group comprises from 3 to 20 carbon atoms; styrene,methylstyrene, ethylstyrene, tert-butyl styrene, chloromethylstyrenevinyltoluene; derivatives thereof and the copolymers of these monomerswith one another and/or with other comonomers, and metal oxide particleschosen from particles of metal oxides of iron, titanium, cobalt, zinc,copper, manganese, nickel; magnetite; hematite, ferrites such asmanganese, nickel or manganese-zinc ferrites; alloys of cobalt, nickel.18. The complex as claimed in claims 15 and 16, characterized in thatthe envelope polymer is chosen from at least one hydrophilic homopolymeror copolymer chosen from homopolymers or copolymers: derived from thepolymerization of at least one monomer chosen from monomers derived fromacrylamide or from methacrylamide; acrylic acid, methacrylic acid;acrylate and methacrylate derivatives; allylamine; styrene derivatives;on the condition, if it is a homopolymer, that this homopolymercomprises ionizable functional groups; in particular copolymers orhomopolymers of maleic anhydride and homopolymers or copolymers ofacryloxy succinimide, polysaccharides, such as chitosan andpoly(galacturonic acid), polypeptides, such as polylysine andpolyarginine, linear or branched polyethyleneimine, and dendrimers. 19.The complex as claimed in claim 18, characterized in that the envelopepolymer is poly(maleic anhydride vinyl ether),poly(N-vinylmorpholine-N-acryloxysuccinimide) orpoly(N-vinylpyrrolidone-N-acryloxysuccinimide).
 20. The use of a complexas defined in claims 15 to 19, for the transfer and/or transport and/orstorage of infectious agents, in particular of a virus and/or bacteriumand/or yeast.
 21. A reagent for extracting and/or identifying and/ordetecting and/or quantifying proteins and/or protein and nucleic acidassociations, characterized in that it comprises, inter alia, a complexas defined in claims 15 to
 19. 22. A reagent for extracting and/oridentifying and/or detecting and/or quantifying proteins and/or proteinand nucleic acid associations, characterized in that it comprisescolloidal particles defined in any one of claims 1 to 4, which make itpossible to obtain a complex as defined in either of claims 15 and 16.23. The reagent as claimed in claim 21 or 22, characterized in that italso comprises at least one means for identifying and/or detectingand/or quantifying said proteins or said nucleic acids, in particular atleast one monoclonal or polyclonal antibody for identifying and/ordetecting and/or quantifying at least one protein, and at least oneprimer, preferably at least two primers, and/or at least one nucleotideprobe, preferably at least two nucleotide probes, specific for at leastone nucleic acid, for the identification and/or detection and/orquantification thereof.
 24. A vaccinal composition, characterized inthat it comprises, as active principle, at least one complex as claimedin claims 15 to 19, optionally combined with a pharmaceuticallyacceptable vehicle and/or excipient and/or adjuvant and/or diluent. 25.A pharmaceutically acceptable vehicle for a vaccinal composition,characterized in that it consists of a particle comprising a core and anenvelope as defined in any one of claims 1 to 4.